Thus, the use of CreERT2 is more preferred in several biological fields. Cre-mediated recombination occurs between one of the two identical pairs of lox sites (a pair of loxP sequences and a pair of lox2272 sequences) in a mutually exclusive way in the Brainbow system [].Moreover, excision by one recombination event removes a lox site, To identify LoxP insertions, as a general practice at all centers, the two target sites were amplified individually to look for an increase in the amplicon size, which occurs if LoxP sites are inserted successfully. Tonna S, Takyar FM, Vrahnas C, Crimeen-Irwin B, Ho PW, Poulton IJ, Brennan HJ, McGregor NE, Allan EH, Nguyen H, Forwood MR, Tatarczuch L, Mackie EJ, Martin TJ, Sims NA. Inclusion in an NLM database does not imply endorsement of, or agreement with, 2012). This system includes digestive tract and various accessory organs to assist digestion by secreting enzymes. Evidence for multiple promoters and for regulation by DNA methylation. Gu H, Zou YR, Rajewsky K. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. RNA-guided human genome engineering via Cas9. The in vivo use of the Cre/lox system involves both mice expressing Cre enzyme and mice with loxP sites inserted at a selected trans-genic or endogenous locus of interest. Primer pairs for loxP4 (loxP4A F/R) and loxP5 (loxP5B F/R) were used for amplification of control DNA samples. Cebrian C, Asai N, D'Agati V, Costantini F. The number of fetal nephron progenitor cells limits ureteric branching and adult nephron endowment. Tronche F, Kellendonk C, Kretz O, Gass P, Anlag K, Orban PC, Bock R, Klein R, Schtz G. Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Following in vitro transcription, RNA was purified by ethanol precipitation with 1/10 volume of 3M sodium acetate. We hypothesize that smaller oligonucleotides, or oligonucleotides generated from a different source, potentially a sequenced plasmid, might decrease the frequency of error and increase the correct targeting allele efficiency. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. Targeting Cre recombinase to specific neuron populations with bacterial artificial chromosome constructs. Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis. The nestin (Nes) is also expressed in precursor cells of neuron and glia [32]. . It is unclear whether this was due to location of injection (cytoplasmic vs. pronuclear), variation between injection days, or was affected by the distance between the Cas9 cleavage sites. The loxP site is a 34 bp sequences consisting of a two 13 bp inverted and palindromic repeats and 8 bp core sequences (Figure 1A). LoxP4 insertions were observed in 10/47 mice (21%), loxP5 insertions were present in 11/47 mice (23%), and insertion of both loxP4 and loxP5 occurred in 11% (5/47) of the N0 mice from the Dock7 cKO1-1,-2 injection (Table 3). Uroplakin 2 (Upk2) is expressed in urothelium of the bladder and used for the bladder cancer and urothelium functional studies [107]. DNA was precipitated with an equal volume of isopropanol, and pelleted by centrifugation. Conditional knockout mice are then generated by breeding the Cre-driver strain with a floxed mouse strain (Figure 1B). . Tallquist MD, Weismann KE, Hellstrm M, Soriano P. Early myotome specification regulates PDGFA expression and axial skeleton development. Interestingly, even with a small group of six targeted alleles, the success rate observed in this study recapitulated that observed by the KOMP repository of 50% [KOMP repository germline transmission rates and (Cotton et al. Furthermore, additional issues that prevented generation of the Dock7 transgenic line from the KOMP-targeted cells included an incomplete targeting cassette and a mixed population of ES cells from the parental clone (Ryder et al. However, no large deletions or insertions in the loxP site were observed in the oligonucleotide donors. A representative example of six subclones is shown, which reflects the amplification patterns found within the subclones. WebCre aids in the targeted transgenesis of the DNA gene segment of interest, as it supports the mapping of the transgene insertion sites, known as loxP sites. *Table 2 is based on the in situ hybridization data of the adult mouse brain (postnatal 56 day). The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimaerism. In the study of oligodendrocytes, proteolipid protein (myelin) 1 (PLP1) promoter is very useful in the studies of the gene function in myelination of mature oligodendrocytes [35]. To generate the spatiotemporally controlled mutant mice, two elements are needed in the Cre-loxP system [10,11,12,13]. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. As a negative control, off-target activity was assessed in the wild-type C57BL/6J mouse at the insertion sites for both loxP4 and loxP6, as shown in Figure S4. . Gossen M, Freundlieb S, Bender G, Mller G, Hillen W, Bujard H. Transcriptional activation by tetracyclines in mammalian cells. Two corresponding oligonucleotides were synthesized with 7980bp homology arms flanking the loxP sequences. Lysozyme 2 (Lyz2) is expressed in various myeloid cells including monocytes, macrophages and granulocytes. Transgene - Wikipedia Several of the loxP sites sequenced contained small 1- to 2-bp deletions or 1-bp substitutions, whereas some other insertions carried larger deletions within the loxP site itself (Figure 5). The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Rubtsov YP, Niec RE, Josefowicz S, Li L, Darce J, Mathis D, Benoist C, Rudensky AY. The inserted loxP sites and flanking sequences in the N1 and selected N0 mice were cloned and sequenced. The useful Cre-related portal sites and databases will enhance the efficiency of research by allowing researchers to find and obtain the suitable Cre-driver lines for research. A conditional knockout resource for the genome-wide study of mouse gene function. The fused Cre protein is called CreER recombinase, tamoxifen (also known as CreERT, Cre-ERT) and normally presented in the cytoplasm in a form that binds to heat shock protein 90 (HSP90). Leucine-rich repeat-containing G protein-coupled receptor 6 (Lgr6, also known as GPCR) is expressed in hair placodes at embryonic stage and in follicle bulge of dermis at adult stage [50]. Ogilvy S, Elefanty AG, Visvader J, Bath ML, Harris AW, Adams JM. Three-wk-old C57BL/6N female mice were injected with 5IU/mouse of pregnant mare serum followed 48hr later with 5IU/mouse of human chorionic gonadotropin, and mated to C57BL/6N males. DNA fragment size of the loxP (L), wild type (+), and small deletions due to Cas9 cleavage () are indicated. The DNA pellet was washed with 750l of 70% EtOH, air dried, and resuspended in water. However, the insertion reaction is more difficult It (A) One sgRNA was validated within intron2 and intron4, a distance of 2.1kb apart. Founder 142 did not produce germline transmission of single inserted loxP site after 28 pups or four litters; since founder 142 genotyped positive for only one loxP site, breeding was discontinued. Schofield PN, Hoehndorf R, Gkoutos GV. A mouse informatics platform for phenotypic and translational discovery. Differential oocyte-specific expression of Cre recombinase activity in GDF-9-iCre, Zp3cre, and Msx2Cre transgenic mice. Rajewsky K, Gu H, Khn R, Betz UA, Mller W, Roes J, Schwenk F. Conditional gene targeting. Primers used for genotyping are indicated, with the expected product size. Bullard T, Koek L, Roztocil E, Kingsley PD, Mirels L, Ovitt CE. Analysis of the Dock7 first knockout allele (Dock7tm1a). To detect lox (including loxP and its mutants, lox66 and lox71) insertion in blastocysts (Fig. Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice. Mice were scored as having an intact cassette if a product was obtained from each of the three primer pairs: neo-loxP2A F/R, neo-loxP2B F/R, and loxP3 F/R. Table 1 shows the clones injected, number and sex of chimeras obtained, and success in germline transmission of each allele. Leptin action on GABAergic neurons prevents obesity and reduces inhibitory tone to POMC neurons. Care must be taken to avoid disrupting the regulatory sequences in 5- and 3-untranslated A majority of the skeleton is formed by endochondral ossification. Search for other works by this author on: Corresponding author: Center for Molecular Medicine, Maine Medical Center Research Institute, 81 Research Drive, Scarborough, ME 04074. Shimshek DR, Kim J, Hbner MR, Spergel DJ, Buchholz F, Casanova E, Stewart AF, Seeburg PH, Sprengel R. Codonimproved Cre recombinase (iCre) expression in the mouse. Stability of the regulatory T cell lineage in vivo. The number of offspring (N1) from each of the chimeric mice is shown, along with numbers of mice with agouti coat color and amplification of the targeting cassette. DNA isolated from C57BL/6J mice was used as a negative control (Figure S4). Flanking PCR primers designed to amplify 300600bp fragments are listed in Table S5, Table S6, and Table S7, with the sequence and genomic coordinates of each potential off-target region. LoxP mutations in mice 3 and 116 were passed via germline transmission. Since initial offerings of targeted ES cell clones, the KOMP project has moved forward to the production, gene expression analysis (West et al. However, it is still unclear if variability between mouse strains can affect the overall outcome of success in developing a floxed allele using CRISPR/Cas9. Although the majority of the mutant alleles that have generated mouse strains are correctly targeted alleles, thorough molecular characterization has identified limitations that users need to be of aware of in characterizing their own particular lines of interest. Federal government websites often end in .gov or .mil. WebInsertion of loxP sites into intronless genes, as most claudin genes, can be difficult. All PCR reactions were run on a 3% agarose gel to visualize the products. Chromosomes were visualized at 1000 magnification with light microscopy, photographed, and total chromosomes were counted for a minimum of 10 spreads per slide. Tetreault MP, Yang Y, Travis J, Yu QC, Klein-Szanto A, Tobias JW, Katz JP. In all five mice, two deletion products were identified, and correspond to the deletion of exons3 and 4 of Dock7 gene, similar to what is observed in Figure 4. Also, we finally introduce some available links to the Web sites that provides detailed information about Cre mouse lines including their characterization. Using the CRISPR/Cas9 system as an alternative method to insert loxP sites within the Dock7 locus, two conditional deletion strategies were designed (Figure 2). One inserted loxP sites to flank exons3 and 4 (Figure 2A, cKO1), and the second inserted loxP sites to flank exons3 and 7 (Figure 2B, cKO2). Coleman J L, Brennan K, Ngo T, Balaji P, Graham R M et al. However, the rate at which a correctly sequenced floxed allele in cKO1 was generated and passed to the N1 generation was lower, at 1/47 (2%). Zhang M, Xuan S, Bouxsein ML, von Stechow D, Akeno N, Faugere MC, Malluche H, Zhao G, Rosen CJ, Efstratiadis A, Clemens TL. Multiplex genome engineering using CRISPR/Cas systems. Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. Briefly, morulae were collected at 2.5dpc, injected with 1012 ES cells, and 1216 morulae were divided equally between two uterine horns of a 0.5dpc Swiss Webster female. National Library of Medicine Of the four additional mice that showed evidence of insertion of loxP4 and loxP5, two were found to have mutated loxP sites, one died prior to weaning, and the final mouse had no germline transmission of either loxP site after four litters. Chimera1 showed evidence of germline transmission of only a partial Dock7tm1a cassette, while chimera5 produced four agouti offspring, genotyping showed no evidence of the targeting cassette in any of the pups. Lymphocyte protein tyrosine kinase (Lck) is mainly expressed in T cells and used for the selection and maturation studies of T cell development in thymus [63,64]. Targeting a gene with two loxP sites is both time and labour intensive. Additional evidence of mosaicism could be observed in the N0 mice containing both a wild-type and loxP positive allele as well as a deletion of DNA between the Cas9 cut sites, such as in the loxP4 genotyping in cKO1 founder 117. The endocannabinoid system controls key epileptogenic circuits in the hippocampus. it was discovered as a 38-kDa DNA recombinase produced from cre (cyclization recombinase) gene of bacteriophage P1 [3,4,5,6]. Aquaporin 2 (Aqp2) is expressed in collecting duct of the kidney and used in nephrological studies [108]. Generation of Floxed Mice by Sequential Electroporation To date, investigators have had success with germline transmission of the targeted clones, providing valuable tools to the research community (Coleman et al. WebHere we reported the generation of Sigmar1 conditional knockout (Sigmar1 loxP ) mice using CRISPR-Cas9 method to insert loxP sites into the 5'- and 3'-untranslated regions of Sigmar1. 2013). Mice containing the correctly targeted floxed allele. Mutations at loxP insertion sites. 2014; Yang et al. A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines. Forkhead box P3 (Foxp3) is used for studying the lineage stability of regulatory T cells (Treg) [62]. Solute carrier family 6; neurotransmitter transporter, dopamine, member 3 (Slc6a3 also known as DAT; dopamine transporter 1) is expressed in dopaminergic neurons of the midbrain and used for studying drug addiction and Parkinson's disease [37]. Herrera PL, Orci L, Vassalli JD. These ES cells were grown and expanded according to the instructions for each particular line, and prepared for injection into C57BL/6N morulae or blastocysts. The other chimeric mice had evidence of cells containing sequences amplified by primer pair neo-loxP2A F/R, but not the other two target sites. Examples of genotyping for both loxP insertion and deletion of DNA between the Cas9 cut sites are shown in Figure S2. . These loxP mutations were observed in N0, and their respective offspring. Friedel R H, Seisenberger C, Kaloff C, Wurst W. Koscielny G, Yaikhom G, Iyer V, Meehan T F, Morgan H et al. Recently, Cre-loxp system for more precise control has been continuously developed. Chimeric mice generated from the Dock7tm1a ES cells were identified by their agouti coat color, and bred to C57BL/6J mice. Additional clones targeting Dock7 with conditional knockout potential were not available through the KOMP project at the time these experiments were performed. An example of genotyping results with the chimeras and ES cell subclones can be observed in Figure 1. Mice were screened by genotyping for loxP4, loxP5, deletion of DNA between the Cas9 cut sites (deletion of exon34), and coat color. Androgen receptor function is required in Sertoli cells for the terminal differentiation of haploid spermatids. Simpson CL, Patel DM, Green KJ. Observed mutation or substitutions are shown in gray. 2013). Only founder 117 required screening of 19 pups (three litters). ROSA26: The Safe Harbor Locus in the Mouse Genome A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. The amplification patterns matched what was found in the chimeric mice: some clones had all three products (Figure 1B, subclones1 and 6), and some had a product only from primer pair neo-loxP2A F/R (subclones3, 5). The International Knockout Mouse Consortium (IKMC), and participating organizations, are using both targeted For studying the functional regulation of osteoclasts, cathepsin K (Ctsk) promoter is used [75,78,79,80]. . Temporal control of gene deletion in sensory ganglia using a tamoxifen-inducible Advillin-Cre-ERT2 recombinase mouse. Sternberg N, Hamilton D. Bacteriophage P1 site-specific recombination. Lewandoski M, Wassarman KM, Martin GR. Mice 4 and 6 were not bred. Kopp JL, Dubois CL, Schaffer AE, Hao E, Shih HP, Seymour PA, Ma J, Sander M. Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas. Because our focus was on generating a conditional, floxed allele, we did not breed mice containing biallelic disruption of the Dock7 gene to evaluate germline transmission. Zerucha T, Sthmer T, Hatch G, Park BK, Long Q, Yu G, Gambarotta A, Schultz JR, Rubenstein JL, Ekker M. A highly conserved enhancer in the Dlx5/Dlx6 intergenic region is the site of cross-regulatory interactions between Dlx genes in the embryonic forebrain. Use of the Cre/lox Recombination System to Develop a Non
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